1. Field of the Invention
The present invention in the fields of structural biology, immunology and medicine relates to a newly discovered structural characteristic of the gp120 V3 loop and a resultant “rule” or algorithm, that is used in a method for determining whether a subject is infected with Human Immunodeficiency Virus-1 (HIV-1) that expresses selectivity for CXCR4 (or CCR5) chemokine receptors. A positively charged surface patch defined by V3 loop residues 11 and 24 or 25 at the base of the β-strands in the V3 loop and the homologous β2-β3 chemokine hairpin is responsible for CXCR4 receptor selection.
2. Description of the Background Art
A substantial library of structural data has accumulated describing the major surface protein of HIV-1, gp120. gp120 requires interaction with CD4 and chemokine receptors on the surface of target cells in order for HIV-1 to infect cells. The third variable loop (V3) of gp120 is one of the critical HIV-1 regions responsible for initial interaction of gp120 with host cells. The V3 loop interacts with the chemokine receptors [1, 2], and specific V3 residues dictate the choice of co-receptor usage [3-14]. (Numbers appearing between square brackets “[ ]” refer to the numbering of references cited in a list prior to the claims.) HIV-1 co-receptor usage plays a critical role in viral tropism, transmission, and disease progression in infected individuals [15, 16]. Deletion of the V3 loop abolishes virus infectivity [17-19], and replacement of the V3 loop with a portion of the CXC chemokine SDF-1 preserves infectivity [20]. In addition, several human monoclonal antibodies (mAbs) specific for the V3 loop, derived from the cells of HIV-1-infected patients, are broadly neutralizing [21-24], and immunization of animals with proteins into which the V3 loop has been inserted results in HIV-1 neutralizing antibodies [25, 26]. All of these well-documented findings provide evidence that the V3 loop plays a critical role through direct protein-protein interactions in virus infectivity. In addition, X-ray and NMR studies have confirmed that different V3 loops are able to adopt the same overall 3D structure: a β-hairpin [27-33]. Thus, different V3 loops exhibit common protein structural characteristics, and the V3 loop represents a functionally important region of the virus envelope, despite its well-described variation in sequence.
HIV-1 strains that require the CXCR4 co-receptor on target cells for infection are termed “X4”-tropic viruses; those requiring the CCR5 co-receptor are termed “R5”-tropic; those able to utilize both are termed “dual-tropic.” Co-receptor utilization has often been predicted using HIV-1 envelope sequence information, resulting in assignment of viral tropism, or “phenotype”, on the basis of the V3 loop sequence. This prediction has turned out to be less accurate for X4 than for R5 viruses (Resch et al. [35] and is confounded by the use of genotypic analyses in different studies of viral quasi-species or molecular clones. Further confusion results from historical nomenclature because the term “tropism” was originally associated with viral growth characteristics. It is now appreciated, however, that the original designations (“slow/low” vs. “rapid/high” and non-syncytium-inducing (NSI) vs. syncytium-inducing (SI)) do not always correlate with co-receptor usage (E M Fenyö et al., J. Virol., 62:4414, 1988; M. Tersmette, J Virol. 63:2118, 1989; P. Zhong et al. [77]. Thus, any structure/function analysis of HIV-1 tropism must be based on a panel of viruses with carefully defined co-receptor usage.
Mutational data suggested that two positions in V3 sequences were the primary determinants of HIV-1 tropism for X4, as opposed to R5, co-receptors: Mutation of the negatively charged residue at position “25” (based on numbering of the V3 loop in the consensus sequence of HIV-1 subtype B beginning with the N-terminal Cys assigned position 1, i.e.,
 TRPNNNTRKSIHIGPGRAFYTTG IIGDIRQAHC[SEQ ID NO:1]where residues 1 and 25 are shown in bold-face, italics), to a positively charged residue changes the tropism of an R5 virus to that of an X4 virus [10]. Conversely, a negatively charged residue may be accommodated at position 25 in an X4 virus if a positively charged residue is present at the “11” position (underlined above).
These findings gave rise to the so-called “11/25 rule” [3, 10, 13, 34]. According to this rule, if a positive charge is present at position 11 or 25 of the V3 loop sequence, the virus is predicted to be X4-tropic. However, this scheme is not truly a “rule” because of its low predictive accuracy. Additional “rules” have been promulgated in attempts to decipher the basis of receptor tropism through sequence alone, but the best performing algorithms could achieve only about 70% predictive accuracy [13, 35, 36]. Moreover, these computational studies did not rely upon a “gold standard test set” of V3 sequences with exclusive CCR5 or CXCR4 selectivity that were directly verified by assays of co-receptor usage. Finally, the mechanism by which these amino acid substitutions produce a change in tropism remains unknown.
The mechanism of tropism might be better appreciated if the structure of the V3 loop, including the positioning in space of residues 11 and 25, could be observed. Unfortunately, the V3 loop is characterized by structural disorder such that its deletion has been required for crystallographic resolution of gp120 structure [37, 38]. This characteristic has made experimental determination of the native V3 loop structure difficult. The structural variation associated with such disorder, from a statistical mechanics point of view, may range from a complete absence of order (very little chance that any give structural conformation exists at any given time) to a strong, albeit not complete, preference for a single conformation [39]. However, it is often true that “natural” structural disorder in proteins favors a particular conformation in the right environment [40]. In the case of the V3 loop, this favored conformation may be both biologically important and biologically vulnerable.
Ordered structures of the V3 loop in complex with neutralizing mAbs have been resolved by both X-ray crystallography NMR spectroscopy [27-29, 41-44]. Given the neutralizing activities of these mAbs, it may be inferred that the structural conformation of V3 present in the V3/mAb complexes are the same as those that occur when the native virus interacts with the chemokine receptors. Moreover, structural details of the V3 loop in complex with mAbs that neutralize R5- and X4-tropic viruses have recently been obtained. Notably, these structures include the “11” position but not the “25” position of V3. However, the extent of structural detail is adequate to offer insights into how different viral V3 sequences dictate the recognition of different chemokine receptors.
The central portion of the V3 loop is a β-hairpin fold (strand-tum/loop-strand) the N-terminal strand of which makes most of the binding contact with neutralizing antibodies [28, 29]. In the only crystallographic structure of a V3 peptide in complex with an R5-neutralizing human mAb, the N-terminal strand exhibits one specific electrostatic contact (at position 18), but most of the interaction with the antibody is through backbone and non-specific side chain contacts of antibody atoms with the V3 β-hairpin's N-terminal strand [29]. Thus, conservation of the β-hairpin structure may be required to preserve this antigen/antibody interaction. However, since the side chains in a β-strand point in a perpendicular direction from the plane of the β-strand, and the broadly neutralizing mAb studied recognizes primarily main chain and hydrophobic side-chain atoms of the β-strand, a great deal of sequence variation is afforded without impact on the observed binding mode, explaining the broadly neutralizing capacity of the mAb. Indeed, the sequence of this region of the V3 loop is known to be extremely variable [45]. Thus, the crystallographic and NMR data may explain why some sequence variability of the V3 loop may be inconsequential to chemokine receptor binding and to recognition by certain antibodies.
The natural ligands of the HIV-1 co-receptors are chemokines, and the classification of chemokine receptors is based on the grouping of these chemokines. For example, CXCR4 is the receptor for the CXC chemokines (e.g., SDF-1) and CCR5 is a receptor for several CC chemokines (RANTES, MIP-1α and MIP-1β). Chemokines contain a central β-sheet and C-terminal β-helical structural features [46]. Interestingly, a structural homology is evident between the β2-β3 hairpin of both groups of chemokines and the β-hairpin of the V3 loop: The homology “segregates” with biological activity such that (1) an homology exists between the structure of the V3 loop when in complex with an X4-neutralizing antibody and the β2-β3 hairpin in SDF-1, whereas (2) a parallel homology exists between the structure of the V3 loop when in complex with an R5-neutralizing antibody and the β2-β3 hairpin in RANTES, MIP-1α and MIP-1β [28].
Mutational analysis of the chemokines has revealed that a cluster of residues (the “N-loop”) located on the surface of chemokine MIP-1β near the base of the β2-β3 hairpin is one of the important sites for receptor binding [47]. Moreover, as an impetus for the present study, the present inventors noticed, that two residues in or near the β2 and β3 strands that are close to the N-loop in 3D space may align with the so-called “11” and “25” residues of the V3 loop which play a critical role in determining chemokine receptor selection. The inventors therefore used the known 3D structures of the chemokines and of the V3 loops of both R5 and X4 viruses, along with a library of V3 loop sequences from primary HIV-1 strains with confirmed (and exclusive) CXCR4 or CCR5 usage, to elucidate the structure/function relationships that determine chemokine receptor selectivity and therefore HIV-1 tropism.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.